The present invention relates to a method for immobilizing biomolecules on a conductive substrate. More particularly, this invention relates to a method for electrodepositing biomolecules such as enzymes and antibodies onto the conductive substrate followed by chemical crosslinking of the biomolecules to form a water insoluble mass.
Immobilization of biomolecules on a substrate is necessary for many commonly employed analytical or industrial applications utilizing biomolecules. Immobilization converts typically water soluble biomolecules such as enzymes or antibodies into water insoluble complexes through attachment of at least some of the molecules to a water insoluble physical support material. Biomolecules so immobilized can often be used in many applications without change in their concentration or activity over time. Immobilization has an additional advantage in that biomolecules can be retained only in specific desired regions of an apparatus or sensor, minimizing material costs and maximizing detectable bioactivity.
An important analytical use of immobilized biomolecules such as enzymes, antibodies, or glycoproteins such as lectins is in biosensors that detect the presence or concentration of selected physiological molecules as a result of the interaction of the physiological ligand with the immobilized biomolecules. Unfortunately, adapting known immobilization techniques for use in conjunction with the miniature electrochemical biosensors fabricated using conventional semiconductor manufacturing techniques can be difficult. Current semiconductor techniques allow fabrication of a sensor with a surface area of as little as 1.0 .mu.m.sup.2. This small areal dimension presents difficulties in sensor construction since the fabrication of biosensors generally requires deposition of a biomolecule only on the surface of the working electrode, which is used to monitor a product of the enzymatic reaction. Biomolecules deposited on other areas of the biosensor would react to produce a product that could not be detected, wasting the often costly biomolecule.
One method of overcoming problems in depositing biomolecules relies on electrophoresis to promote migration of charged biomolecules such as proteins, amphipathic lipids, or nucleic acids. In the appropriate medium, such biomolecules contain positively or negatively charged moieties that are attracted to an opposing pole of a generated electric field. Migration of the biomolecule contained within the medium toward and deposition on an electrode having a polarity opposite that of the charged molecule is therefore promoted if a potential across two electrodes in a medium is created.
For example, electrophoretic deposition techniques were utilized by Ikariyama et al., J. Electrochem, Soc., 136(3), pp. 702-706 (1989). This method promoted the codeposition of platinum particles and the enzyme glucose oxidase. At the pH where platinization normally takes place (pH=3.5), the glucose oxidase has a net positive charge since the pH is lower than its isoelectric point of 4.3. The glucose oxidase is therefore attracted to an oppositely charged electrode along with the platinum ions. To enhance the stability of the deposited enzyme, the sensor is dipped into solutions of albumin and glutaraldehyde to cross-link the molecules. However, this technique can result in the inactivation of the enzyme because of the relatively low pH employed to promote codeposition of enzyme.
Aizawa et al., J. Chem. Soc. Japan (Nippon Kagaku Kaishi), 11, pp. 2210-2213 (1987) have reported the development of a technique for electrodepositing glucose oxidase. The glucose oxidase was absorbed on a platinum electrode surface at a controlled potential from an aqueous solution. The optimal conditions for their deposition were 0.1 V vs. Ag/AgCl and pH 3. The maximum thickness achieved by this method was four molecule thick layers of glucose oxidase, equivalent to approximately 56.times.10.sup.-9 m maximum glucose oxidase film thickness. The technique was evaluated at pH values ranging from 3 to 9 resulting in the optimal deposition at a pH of 3. The extremely thin layer deposited by this technique is inadequate for many Purposes that require that the interaction of the ligand with the biomolecule be a non-rate limited factor in the reaction. A further disadvantage is the limited lifetime and reusability of such thin layers of immobilized molecules.
Accordingly, it is an object of this invention to provide a method for depositing and immobilizing a relatively thick layer of molecules biomolecules on a conductive substrate so that the interaction of a ligand with a biomolecule is not rate-limited.
It is another object of this invention to provide a method for reproducibly and accurately depositing and immobilizing biomolecules onto miniature electronic sensors.
A further object of this invention is to provide a method for simultaneously depositing and immobilizing a plurality of different biomolecules onto a conductive substrate.
An additional object of this invention is to provide a method for depositing and immobilizing biomolecules in a manner resulting in layers with thicknesses ranging from 10.sup.-8 to 10.sup.-5 m.
Accordingly, a method for preparing a biosensor electrode having a conductive substrate coated with a desired biomolecular species includes preparing a solution of at least one species of biomolecule intended for deposition buffered within a range sufficient to prevent denaturization of the biomolecules, and to a value such that all species of biomolecules intended for deposition have the same net electric charge sign due to their respective isoelectric points. The biosensor electrode and a counter electrode are immersed in the solution, and a potential difference of between about 100 millivolts and 1 volt is created between the electrodes, causing a current to flow. The potential is varied with time in a manner calculated to cause the current flow between the electrodes to remain substantially constant, inducing migration toward and subsequent deposition on the biosensor electrode. After a desired thickness of the biomolecule has been deposited on the biosensor electrode, the stability of the deposited biomolecular film can be enhanced by crosslinking with a suitable crosslinking agent.
In preferred embodiments the solution containing a biomolecule is aqueous, and the current between the biosensor electrode and the counter electrode is selected to be about 5 mA/cm.sup.2. The voltage is adjusted upward over the course of the deposition process to maintain a constant current despite the increasing electrical resistance of the biosensor electrode due to biomolecular film deposition. Deposition continues until an average film thickness over the biosensor electrode is greater than 5 micrometers.
One advantage of the present invention is its wide applicability to conductive substrates having differing geometries. Because biomolecule deposition is electrophoretically based, biomolecules can be deposited on virtually any conductor or semiconductor independent of shape or topography. For instance, a very rough conductor surface may be uniformly covered with a biomolecular film or the inside of a conductive material such as platinum tubing could be coated with a biomolecule very easily and inserted into a flowing stream system for analytical purpose. Also, in contrast to many other film deposition techniques, the deposition of the biomolecules is localized in such electrophoretic techniques, with the desired biomolecule only being deposited in the vicinity of the conductive substrate.
Another advantage lies in the relatively thick layers of biomolecules which can be deposited on a biosensor electrode. Many physiologic applications involve interaction between relatively large amounts of a particular physiologic ligand to be detected and a biomolecule deposited on a biosensor electrode. Very thin monomolecular films or films having a thickness much less than 1 micrometer may cause the reaction between the physiologic ligand and the biomolecule to be rate limited due to the small amounts of biomolecule available for reaction, decreasing the sensitivity of the sensor and providing a non-linear interaction function (between the biomolecule and the ligand) that could make determination of the amount of ligand present in a solution difficult.
Another advantageous feature of the present invention results from the application of a constant current over the course of the deposition on the biosensor electrode. This results in a steady deposition of the biomolecule on the biosensor electrode even though biomolecule deposition blocks the electrode surface and ultimately increases electrical resistance. Because the resistance of the biosensor electrode increases over the course of the deposition process, both the current and the rate of biomolecule deposition ordinarily decreases unless the voltage is not gradually increased. This decrease in deposition rate would continue until no further deposition occurs, even though the deposited film may be much less thick than required. In contrast, the present invention holds the current constant and allows the voltage to increase as resistance increases, until the voltage approaches a value at which water will be either reduced or oxidized to form gas bubbles, thereby promoting formation of films having a thickness greater than 10 micrometers.
Another advantage of the present invention is its utility at pH values comparable to the physiologic pH values at which a biomolecule is formed. Very thick films of proteinaceous biomolecules such as enzymes can be deposited at physiologic pH (7.4) in aqueous solutions, minimizing potential problems with inactivation or denaturation of the enzyme that can be encountered with other methods of depositing enzyme films.
Additional features and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.